DESCRPITION (provided by applicant): Mucin-type O-linked glycosylation is a post-translational modification in which proteins are highly decorated by carbohydrate chains. These oligosaccharides serve as mediators of information between cells and are crucial for cell-cell communication. Changes in expression patterns of mucin-type O-linked glycoproteins is believed to contribute to both the invasive and metastatic properties of tumor cells. The biosynthesis of mucin is initiated by a family of enzymes collectively known as the ppGaINAcTs. Characterization of ppGaINAcT substrate specificity and the properties conferring this specificity for each individual isoform has been a difficult task. We propose to use a unique combination of chemistry and molecular biology to facilitate the further understanding of this enzyme family in a stepwise process. Aim 1. Synthesize and systematically analyze a library of glycopeptides to determine the substrate preference of each ppGaINAcT isoform. Aim 2. Create chimeric enzymes to determine what parts of the protein are responsible for conferring specificity. Finally, Aim 3. To correlate the specificity patterns for various isoforms observed in vitro with substrate preference within cells.